The proposed research involves the development and study of active-site-directed inhibitors of several of the key enzymes of coagulation and fibrinolysis, i.e., thrombin, plasma kallikrein, plasmin, and urokinase. The investigation will be directed towards the following goals: 1. Elucidation of the factors determining the specificity and the mechanism of action of the enzymes. 2. Selective suppression of the enzymes to delineate their role in the overall clotting and fibrinolytic processes. 3. Attainment of clinically useful anticoagulant and antifibrinolytic agents. 4. Improved enzyme purification procedures by the use of specific and highly active inhibitors as affinity chromatography ligands. The inhibitory compounds to be devoted to goals 1-3 will consist of amidino-substituted derivatives of aromatic and heterocyclic ring structures and are an outgrowth of our previous extensive experience with synthetic inhibitors of arginine- and lysine-specific esteroproteases. The compounds are conceived along two lines: a. As transition state analogs designed to form a tetrahedral adduct at the active site. b. As trifunctional compounds, capable of establishing extended contact with the active site beyound the confines of the specificity pocket. The potency of the inhibitors will be determined by in vitro assays employing synthetic and natural substrates and also by in vivo testing in experimental animals. The ligands for goal 4 will be derived from amidinoindole and from aromatic diamidines, i.e., compounds of proven high affinity and selectively for the enzymes under consideration. The inhibitors will be furnished with an amine-bearing side chain for covalent bonding to the column material. The ability of the affinity matrices to discriminate between related enzymes will be further augmented by the use of potent and specific synthetic inhibitors in the elution process.